AMPLIGEN® (rintatolimod) poly(I):poly(C12U) was developed as a synthetic double-stranded ribonucleic acid (dsRNA) for therapeutic applications based on an understanding of both the beneficial and adverse effects induced by poly(I):poly(C) on the physiology of a subject. Acting on the hypothesis that the nucleotide sequence requirements for beneficial and adverse effects are different, poly(I):poly(C12U) was developed by us to preserve the beneficial aspects of dsRNA without the adverse effects of poly(I):poly(C) by modifying the latter's structure with the occasional introduction of uridylate into the poly(C) strand to produce duplexes containing specifically-configured regions which are not base paired (i.e., “mismatched”) at the position of the modification. These regions accelerate dsRNA hydrolysis and lessen toxicity (Greene, 1984). On the other hand, the ability to induce interferon synthesis was retained as long as the modified dsRNA persisted for a half life of at least five minutes and the frequency of random insertion into the poly(ribocytidylic acid) strand was not greater than each 0.5 to 1.0 helical turn of perfectly base-paired dsRNA (Brodsky, 1987).
While poly(I):poly(C12U) is stable in solution, it is susceptible to hydrolysis like all other conventional nucleic acids. The hydrolysis is highly dependent on nucleic acid structure, as well as on the presence of nuclease and divalent cations, pH, and temperature. RNA is more susceptible to hydrolysis than DNA because of the 2′-OH group present in the former that facilitates hydrolysis. Moreover, poly(I):poly(C12U) was designed to degrade more rapidly than other dsRNA in a nuclease-containing environment, such as blood and other tissue fluids. Nucleic acids are initially stable in physiological salt buffers at room temperature, but gradually begin to degrade with time. This hydrolysis rate is temperature dependent, increasing greatly at higher temperatures.
Properties of poly(I):poly(C12U) are characterized by physico-chemical assays as shown in Table 1. Circular dichroism (CD) (e.g., ellipticity, melting behavior) is used to characterize the double-helical RNA structure, which is critical to potency. Briefly, Toll-like receptor 3 (TLR3) is activated by dsRNA (Alexopoulou, 2001), which leads to a host defense recruitment sequence, ultimately producing type I interferons (Schroeder, 2005). Initiation of interferon production by dsRNA binding to TLR3 requires RNA helical structure (Bell, 2006). Although X-ray diffraction and NMR alone are the definitive techniques to determine RNA second-order structure, CD measurement with a combination of scanning and thermal stress modes also can provide precise characterization of the critical double-helical structure. Indeed, minor changes in second-order structure of polynucleotides have been measured by CD (Gray, 1995), including the effects of ligand binding (Sumita, 2005).
TABLE 1Biological Activity and Measured Attributes.Measured PropertyIdentity AttributeActivity AttributeConformation: Second DegreeCD: EllipticityDouble-Stranded RNA:binding to TLR3integrity of helixCD: Melting Behavior:Double-Stranded RNA:binding to TLR3Melting Point ½ Widthintegrity and uniformityof helixComposition and SizeMaximum SizeNo. of Repeat UnitsHalf Life: safetyC:U RatioidentityHalf Life: safetyTherefore, circular dichroism can be employed to characterize the therapeutic potency of specifically-configured dsRNA including poly(I):poly(C12U).
As regards adverse toxic effects, the half life of poly(I):poly(C12U) was reduced to a safe level of about 4 to 5 minutes by precise substitution of the poly(C) strand, specifically the cytidine to uridine ratio (U.S. Pat. No. 5,258,369). Introduction of the unpaired base uracil into the poly(C) strand at a ratio of 1:12 (Greene, 1978) resulted in a minimum base-paired length of about one helical turn, which is required for the interaction of dsRNA with its bioactive receptor. Furthermore, placing a maximum size limitation of about 350 repeat units on the dsRNA resulted in a half life of about 4 to 5 minutes (Greene, 1978; Pitha, 1972).
It was our objective to identify a new family of improved dsRNA having specific physico-chemical structure and highly specific biological activities, which includes acting as a selective agonist for TLR3. Its rugged structure as measured by physico-chemical techniques is resistant to molecular unfolding (i.e., denaturation). Improvement in at least one or more biological activities may result from the rugged structure of this particular form of poly(I):poly(C12U). Other advantages and improvements are described below, or would be apparent from the disclosure herein.